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J. Biol. Chem., Vol. 283, Issue 13, 8591-8600, March 28, 2008

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Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis^*

Jonathan C. Gauntlett¹, Susanne Gebhard, Stefanie Keis, Janet M. Manson², Klaas M. Pos, and Gregory M. Cook³

From the Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, P. O. Box 56, Dunedin 9016, New Zealand and the Institute of Physiology and Zürich Centre for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, Zürich, Switzerland

BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane -helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)₇TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-D-maltoside, and subsequently purified it via Ni²⁺-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.

Received for publication, November 20, 2007 , and in revised form, January 23, 2008.

^* This work was supported in part by a Marsden Grant from the Royal Society of New Zealand (to S. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by an Otago Postgraduate Scholarship from the University of Otago, the Todd Foundation Award for Excellence, and the William Georgetti Scholarship from the New Zealand Vice Chancellor's Committee.

² Dept. of Ophthalmology, Harvard Medical School, and The Schepens Eye Research Institute, Boston, MA 02114.

³ To whom correspondence should be addressed: Tel.: 64-479-7722; Fax: 64-3-479-8540; E-mail: greg.cook{at}stonebow.otago.ac.nz.

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